生长速度快且油脂产率高的湛江等鞭金藻诱变株的筛选

【目的】筛选生长速度快的高产油湛江等鞭金藻诱变株。【方法】利用常压室温等离子体射流诱变技术对湛江等鞭金藻进行诱变,通过96孔板、摇瓶和反应器培养对诱变株进行筛选。【结果】传至第13代,600 m L反应器培养7 d,诱变株IM110020最大比生长速率、细胞密度和油脂产率可分别达0.72 d-1、16 750×104 cells/m L、109.8 mg/(L·d),分别比野生株提高12.5%、20.8%和17.9%。【结论】获得了一株生长速度快且油脂产率高的诱变株IM110020,且经过多次传代性质稳定。     

微拟球藻富油藻株筛选及柱状光生物反应器培养评价研究

研究以亲脂性荧光染料BODIPY505/515和流式细胞仪为基础, 从多株诱变海洋微拟球藻(Nannochloropsis oceanica)中筛选到4株候选富油藻株(MT-1,2,3,4), 并利用柱状光生物反应器对诱变株的产油能力进行了综合评价。结果表明, 藻株筛选时最佳BODIPY505/515使用浓度为0.87 μg/mL, 染色时间为10min; 4株诱变株产油性能较野生株有较大提高, 其中MT-4油脂积累达到了干重的66%, 油脂产率比野生型藻株提高了45%, 达到了27.32 mg/(L·d)。4株诱变株的脂肪酸组成合适, 其中C16和C18之和占78%以上, 且主要以饱和脂肪酸和单不饱和脂肪酸为主; 多不饱和脂肪酸只占总脂肪酸的6%—8%, 非常适合生物柴油生产。研究提供了一种针对海洋微拟球藻富油藻株快速、有效的筛选方法, 并以此为基础筛选得到4株极具生物柴油生产潜力的候选藻株, 有望用于规模化生产。     

耐高温拟微绿球藻诱变株的筛选与培养

[目的]筛选拟微绿球藻耐高温突变株,提高其高温耐受能力,延长夏季养殖时间。[方法]对拟微绿球藻进行3次EMS重复诱变和反复45℃高温处理后,利用200 mmol/L HYP压力进行48孔板和24孔板两轮筛选,得到的优势突变株进行反应器高温培养评价和夏季室外培养验证。[结果]夏季室外培养,突变株HT39最终生物积累量和EPA含量分别达到1.93 g/L和3.62%,比野生株提高48.0%和21.9%。[结论]利用重复诱变和多孔板压力筛选的方法获得的优势突变株HT39夏季室外培养表现出较强的高温耐受性能,为选育重要经济藻株拟微绿球藻耐高温品系提供了方法依据。     

产耐高温谷氨酰胺转胺酶菌株的快速筛选方法

目的:采用亚硝基胍(NTG)诱变结合96孔板高通量筛选方法筛选产耐高温谷氨酰胺转胺酶(MTG)的茂原链霉菌(Streptomyces mobaraensis)。方法:通过优化96孔板高通量测定MTG活性的方法、确定筛选温度和时间,建立了产耐高温MTG菌株的快速筛选方法;通过优化NTG诱变条件建立了筛选突变库;通过96孔板高通量初筛、摇瓶复筛获得了产耐高温MTG的突变株12-82,并通过摇瓶发酵对12-82所产MTG进行热稳定性分析。结果:采用2mg/ml NTG、p H8.0、60min的诱变条件获得突变株,将突变株的发酵上清液于70℃水浴7.5min,再在37℃空气浴、反应10min的条件下测定MTG活性,从5 200株突变株中筛选出5株产耐高温MTG的突变株,其中突变株12-82在50℃水浴60min以及70℃水浴1.5min的酶活残留率均比出发株高出近20%,且80℃保温2min仍有11.9%的酶活残留率。结论:利用NTG诱变结合96孔板高通量筛选的方法筛选到5株所产MTG热稳定性相对较高的突变株,其中突变株12-82在50℃、70℃和80℃的酶活残留率均有10%~20%的提高。这为高温食品加工领域所需耐高温MTG生产菌株的高效筛选提供了可行性方案。     

利用叶绿素荧光快速筛选紫外诱变高含油小球藻

本实验利用尼罗红荧光与叶绿素荧光比值表征小球藻的油脂含量,快速筛选出一株紫外诱变高产油藻株。经过叶绿素荧光分析表明,诱变株M2的生长及光合作用没有显著变化。检测诱变株M2总油脂含量32.1%,比野生藻株的23.1%提高了9个百分点。     

三角褐指藻的诱变育种及产EPA的条件研究

旨在获得不饱和脂肪酯EPA产量更高的藻种,利用0.6% EMS对三角褐指藻进行诱变。通过单细胞分离技术得到1株突变株EP1,其EPA产量比出发藻株提高了19.81%。结果显示,突变藻株产EPA的最适条件为:NaNO3 75 mg/L,pH7.5,昼夜温度17-15℃,接种量为12%。最适条件下培养7 d,其EPA产量可达26.77 mg/g。传代试验表明,突变藻株具有较好的遗传稳定性。     

雨生红球藻的紫外、激光复合诱变育种

用紫外线和激光复合诱变生产虾青素的雨生红球藻,以适宜条件下的生长速率和亚适宜条件下的虾青素累积能力为筛选指标。结果表明,与原始出发株比,紫外线诱变后,色青累积速率提高37.8％,但生长速率有所下降,紫外线,激光复合诱变结果,生长速率提高11.1%,培养1个月时虾青素累积量提高52.2%。电镜观察结果表明,激光可刺激叶绿体发育,从而改善了紫外线诱变后的生长抑制状况,展示复合诱变是筛选高产虾青素藻株的有效方法。     

菊粉酶产生菌的筛选及60Co诱变选育简

目的：从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法：通过稀释平板涂布法分离微生物;利用^60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果：分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行^60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46．62U/mL,是未诱变菌株酶活的2．72倍。结论：经诱变得到1株高产菊粉酶活力的突变菌株。     

一株高蛋白含量小球藻TX的分离鉴定及其诱变育种

【背景】小球藻由于蛋白含量高、营养丰富,在水产养殖上可直接作为鱼、虾、贝类的优质饵料。【目的】对从养殖环境中分离的小球藻进行诱变,选育生长快、蛋白含量高的突变株,为水产养殖天然饵料生产提供优良藻种资源。【方法】以从养殖环境中筛选的生长相对较快且蛋白含量较高的TX作为出发藻株,对该藻株进行分子鉴定,并对该藻株进行紫外诱变、甲基磺酸乙脂(ethyl methyl sulfonate,EMS)诱变和复合诱变,采用96孔板高通量筛选技术和递进式重复筛选方法选育高生物量、高蛋白突变株。【结果】经18SrRNA基因序列分析,TX鉴定为Chlorella sorokiniana,从540个可能的突变株中筛选到8个遗传稳定且生长较快的突变株,其中H10的总蛋白含量达64.2%,可溶性蛋白含量达0.44g/L,干重达0.72g/L,分别较出发藻株提高3.4%、15.8%和26.2%。【结论】突变株H10蛋白含量高且生长较快,可用于天然饵料生产。     

集胞藻PCC 6803高产精氨酸藻株的紫外诱变选育

精氨酸在医药和食品工业上具有广泛用途。集胞藻PCC 6803是单细胞蓝藻, 能利用工业废气(主要成分是氮氧化物NO_x)与水反应生成的硝酸盐和亚硝酸盐合成氨基酸等化合物, 因而选育高产精氨酸藻株, 不仅能提高精氨酸产量, 而且能去除工业废气中的NO_x, 具有潜在的应用前景。研究在集胞藻PCC 6803中利用紫外诱变, 筛选抗0.8 g/L D-精氨酸和抗0.2 g/L 6-氮尿嘧啶的突变株, 选育到了一株精氨酸产量显著提高的突变株#13807-111-55, 它每OD₇₃₀值细胞的胞外精氨酸产量相比出发株提高了62.3倍, 达到(0.76±0.1) mg/(L·OD₇₃₀), 总精氨酸产量相比出发株提高了6.0倍, 达到(0.82±0.08) mg/(L·OD₇₃₀)。该突变株每OD₇₃₀值细胞的胞外精氨酸产量明显高于胞内, 表明该突变藻株是精氨酸分泌型, 因而具有潜在的应用前景。     

Isolation of state transition mutants of Chlamydomonas reinhardtii by fluorescence video imaging

Oxygenic photosynthetic organisms adapt to varying light conditions by changing the distribution of light energy between Photosystem II (PS II) and photosystem I (PS I) during so-called state transitions. To identify the genes involved in this process, we have exploited a simple chlorophyll fluorescence video-imaging technique to screen a library of nuclear mutants of Chlamydomonas reinhardtii for colonies grown on agar plates that are disturbed in their ability to regulate light energy distribution between PS I and PS II. Subsequent modulated fluorescence measurements at room temperature and 77 K fluorescence emission spectra confirmed that 5 mutants (0.025% of total number screened) were defective in state transitions. [³²P]orthophosphate phosphorylation experiments in vivo revealed that in one of these mutants, designated stm1, the level of LHC II polypeptide phosphorylation was drastically reduced compared with wild type. Despite WT levels of PS I and PS II, stm1 grew photoautotrophically at reduced rates, compared with WT especially under low light conditions, which is consistent with an important physiological role for state transitions. Our results highlight the feasibility of video imaging in tandem with mutagenesis as a means of identifying the genes involved in controlling state transitions in eukaryotic photosynthetic organisms.     

基于ARTP诱变和高通量筛选的绿针假单胞菌GP72育种方法

【目的】绿针假单胞菌GP72是一种植物根围促生细菌,其分泌的次级代谢产物2-羟基-吩嗪(2-OH-PHZ)具有广谱抗真菌活性,但其产量较低,不能满足农业生产的应用需求,因此需对GP72进行改造,从而提高产量。【方法】从GP72的野生株出发,首次将2-OH-PHZ合成途径的限制性因子Phz O用绿色荧光蛋白(GFP)替换,以一种新型的常压室温等离子体技术(Atmospheric and room temperature plasma,ARTP)进行诱变,通过酶标仪测定96孔板中突变株的荧光强度进行高通量筛选;最后将荧光强度高的菌株中绿色荧光蛋白(Green fluorescent protein,GFP)替换为Phz O以获得2-OH-PHZ高产突变株。【结果】经过五轮诱变后,获得一株荧光强度增加1.62倍的突变株,用phz O基因回替后,该突变株在KB培养基中摇瓶培养时2-OH-PHZ的产量为野生型的4.62倍。【结论】基于安全、高效ARTP诱变技术,并以GFP替换限制性因子作为标记进行高通量筛选,可以快速获得高产2-OH-PHZ的GP72突变株,克服了传统诱变育种方法筛选难度大、费时费力的不足,为其它微生物的育种提供了参考。     

解淀粉芽孢杆菌诱变育种及其突变株在降低酱油中氨基甲酸乙酯的应用

【目的】通过诱变育种提高解淀粉芽孢杆菌JY06利用精氨酸的能力,并将其用于降低酱油中的氨基甲酸乙酯及前体,从而提高酿造酱油的安全性。【方法】采用等离子诱变和紫外诱变两种诱变育种方法对解淀粉芽孢杆菌JY06进行突变,应用高通量筛选手段获得具有高精氨酸利用能力的突变株,验证突变株降低酱油中氨基甲酸乙酯的能力。【结果】获得了12株精氨酸利用能力提高的突变株,与出发菌株JY06相比,突变株C12和E6可使酱油中瓜氨酸含量分别降低了15.6%和14.7%,EC的含量分别降低了19.3%和13.1%。【结论】通过等离子诱变和紫外诱变进一步提高了解淀粉芽孢杆菌JY06降低酱油中EC及其前体瓜氨酸的能力,具有控制或减少酱油中生物危害物的应用潜力。     

阿维菌素生产菌的常压室温等离子体诱变育种及培养基优化

【目的】通过诱变筛选技术选育阿维菌素高产突变株,对其发酵培养基进行响应面优化,提高阿维菌素产量。【方法】采用常压室温等离子体(ARTP)诱变技术,结合链霉抗性和卡那霉素抗性筛选法及96深孔板高通量筛选法,筛选阿维菌素高产株。在单因素实验的基础上,应用响应面分析法对其发酵培养基进行优化,最后确定最佳培养基配方。【结果】获得一株遗传性状稳定的阿维菌素高产株K-1A6,其阿维菌素产量达到4.22 g/L,比出发菌株9-39提高了23.4%,在最佳培养基中阿维菌素产量达到5.36 g/L,较优化前提高了27.01%。【结论】通过对阿维链霉菌9-39菌株进行ARTP诱变筛选及发酵培养基优化研究能显著提高阿维菌素的产量。     

黑曲霉原生质体诱变选育果胶酶高产菌株

通过UV和NTG诱变筛选获得了2株高产果胶酶突变株。以果胶酶产生菌黑曲霉EIM6为诱变材料,采用1．5％的溶壁酶和1．5％的纤维素酶处理其对教生长期菌丝体2h获得高质量的原生质体。采用UV25S或50μg／mL NTG诱变30min,构建原生质体突变库,经刚果红果胶平板筛选获得果胶酶突变株,通过液体深层培养复筛获得高产突变株EIM6-U11、EIM6-N5,酶活力分别从46598．08、46598．08U／mL提高至68596．57、68879．56U／mL,分别提高了47．21％、47．82％。连续8次传代经发酵测酶活力表明高产突变株EIM6-U11、EIM6-N5具有较高的遗传稳定性。     

常压室温等离子体诱变扭脱甲基杆菌AM1高产吡咯喹啉醌

吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)作为一种新型的氧化还原酶辅酶,在医药和食品等领域有广阔的应用前景。为改善扭脱甲基杆菌Methylobacterium extorquens AM1 PQQ生产性能,采用常压室温等离子体(Atmospheric and room temperature plasma,ARTP)进行诱变,结合高通量快速筛选方法,得到以PQQ产量为指标的正向突变株。ARTP诱变的菌株正突变率为31.6%,筛选得到的较优正突变株M.extorquens AM1(E-F3),PQQ产量达到54.0 mg/L,是出发菌株的近3倍。系统的高通量方法筛选ARTP诱变菌为后续进一步提高M.extorquens AM1菌株PQQ的产量奠定了基础,亦为改善菌株生产性能提供了新思路。     

Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9^th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.     

叶绿素合成缺陷型小球藻突变株的选育及其蛋白产能和品质评价

本研究旨在利用常压室温等离子体(atmospheric pressure room temperature plasma, ARTP)诱变技术构建叶绿素合成缺陷型凯式小球藻突变株,筛选出极低叶绿素、适用于发酵生产蛋白质的新藻种。首先经优化诱变处理时间后建立了野生型兼养细胞的致死率曲线,在高于95%致死率条件下处理对数早期兼养细胞,基于可视化藻落颜色变化初筛获得4株突变株。随后在摇瓶中异养培养突变株,系统评价了蛋白生产性能,发现在含有30 g/L葡萄糖和5 g/L NaNO₃的Basal培养基中,突变株P. ks4表现最优,蛋白含量及产率分别为39.25%干重及1.15g/(L·d),氨基酸评分达101.34,叶绿素a含量下降98.78%且不含叶绿素b,含有叶黄素0.62 mg/g而使藻体呈金黄色。本研究为微藻替代蛋白的发酵生产提供了高性能、高品质的新种质P. ks 4。     

Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins

Esterase EstB from Burkholderia gladioli, which belongs to a family of esterases related to beta-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10(6) mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10(5) clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (T(m) was raised by 13 degrees C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.